Genetic variability in stroke patients: CYP2C19 polymorphisms unraveled

Objective To study the distribution characteristics of CYP2C19 polymorphisms in patients suffering from stroke in Han Chinese patients. Method PCR and DNA microarray chip technology were used to detect the CYP2C19 genotype of 549 patients with stroke, and the genotype, allele frequency and metabolic type of patients with different sexes, ages and types of infarctions and the independent risk factors for clopidogrel resistance were analyzed. Results Six genotypes were detected in these 549 patients. A total of 233 (42.44%) patients had the heterozygous allele *1/*2, which was the most prevalent, followed by the homozygous wild-type allele *1/*1 (191, 34.79%). A total of 30 (5.46%) patients possessed the heterozygous allele *1/*3, and 65 (11.84%) patients had the homozygous mutant allele *2/*2. Twenty-nine (5.28%) patients had the compound heterozygous mutant allele *2/*3, and only 1 patient had the homozygous mutant allele *3/*3. The distribution of genotypes, alleles, and metabolic types did not change significantly (P > 0.05) by sex, age, or type of stroke. In addition, no independent risk factors for clopidogrel resistance were found in this analysis. Conclusion The distribution of CYP2C19 genotypes, allele frequencies, and metabolic types in patients with stroke in Han Chinese patients were not correlated with sex, age, or infarction type. The possibilities of hyperglycemia, hypercholesterolemia, hypertriglyceridemia, hypo-HDL-cholesterolemia, hyper-LDL-cholesterolemia and high blood pressure were not statistically associated with CYP2C19 genotypes. CYP2C19 gene polymorphism detection is recommended for patients who are available, and during treatment, the CYP2C19 genotype can be used to guide personalized precise medication use in patients with stroke.


Introduction
Stroke, also referred to as cerebral infarction, is a frequent clinical condition of the brain [8].It is a substantial contributor to disability and the second most common cause of mortality worldwide [25].Moreover, strokerelated death and disability have caused the loss of over 116 million years of healthy life each year [18].Examples of disabilities include lack of muscle coordination, temporary or permanent bodily paralysis on one or both sides, and speech or feeding issues [25].
Stroke mainly affects the brain.Tissues inside the brain are damaged or necrotic brain tissues are formed because of a lack of blood and oxygen supply.
Brain infarction often happens when a clot (ischemic stroke) or a rupture (hemorrhagic stroke) blocks a brain artery [25].Three different forms of stroke exist: emboli, which are produced by blood clots from other tissues,atherosclerosis of the large cerebral arteries (atherothrombotic); and occlusion of perforator arteries (lacunar) [1].Platelet activation and aggregation are the key factors in atherosclerosis and arterial thrombosis.A study revealed that P2Y12 expressed on platelets plays an important role in the activation and aggregation of platelets [20].Therefore, antiplatelet therapy is one of the drug therapies for stroke [12].Aspirin and clopidogrel are the most commonly acceptable options in antiplatelet therapy,aspirin permanently inhibits cyclooxygenase (COX) enzyme activity in the prostaglandin synthesis pathway (PGH2), and clopidogrel works by inhibiting the action of adenosine diphosphate (ADP) on platelet receptors [3,13,21].
Currently, clopidogrel is commonly used in antiplatelet therapy in the Chaoshan area, and many patients are LoFA carriers.Unfortunately, this aspect of research is still relatively unexplored.This investigation sought to examine the outcomes of CYP2C19 genotype identification in stroke patients in Han Chinese, analyze the genotype distribution and provide a theoretical basis for individualized precision treatment.

General data
A total of 549 Han Chinese patients with stroke admitted to the First Affiliated Hospital of Shantou University Medical College between July 2016 and August 2021 were included in this study.These individuals varied in age from 25 to 90 years old.The inclusion criteria were as follows: patients were compliant with the Chinese Guidelines for the Diagnosis and Treatment of Acute Ischemic Stroke 2018 [27], were born in three cities of Chaoshan (Shantou, Chaozhou and Jieyang), were not related to each other, and were Han Chinese.This study was approved by the Ethics Committee of the First Affiliated Hospital of Shantou University Medical College.

Data collection
At admission, the patients' age, sex, infarction type, blood glucose, lipids, triglycerides, High-Density Lipoprotein, Low-Density Lipoprotein and blood pressure were noted.Approximately 2 ml of venous blood was drawn from each patient into a sterile blood collection tube with EDTA anticoagulant.

Reagents and instruments
The nucleic acid extraction reagent was purchased from Xiamen Zhishan Technology Co., Ltd. and was used for extraction on a LabAid820 automatic nucleic acid extraction instrument.The CYP2C19 genetic testing kit, BR-526-24 automatic hybridizer and BE-2.0 biochip reader instrument were purchased from Shanghai BaiO Technology Co., Ltd.The BIOER PCR amplification apparatus was purchased from Hangzhou Bori Technology Co., Ltd.
CYP2C19 genotype testing DNA microarray analysis and polymerase chain reaction were used to analyze CYP2C19 gene polymorphisms.The testing method was as follows: 2 ml of blood sample was collected in an ethylenediaminetetraacetic acid (EDTA) anticoagulant tube on the day while the patient was fasting on an empty stomach.The samples were then stored in a refrigerator at 4 °C, and the DNA was extracted within 24 hours.The PCR mixture, Taq enzyme and DNA template were proportioned using the CYP2C19 gene detection kit (DNA microarray method) provided by Shanghai Biou Technology Co., Ltd.Amplification was performed using the BIOER PCR amplification apparatus as follows: 50℃ 5min, 94℃ 5min, 35 cycles (94℃ 25sec, 48℃ 40sec, 72℃ 30sec), 72℃ 5min.The reagent was prepared according to the instructions.The chip was then removed and hybridized for color development on the BR-526-24 automatic hybridizer.Chip scanning was performed on the BE-2.0 biochip reader, and genotype image analysis was performed using CYP2C19 genotype analysis software.

Observation indicators
Patients suffering from stroke in the EM group, IM group, and PM group were analyzed for genotype and CYP2C19 allele frequency.

Statistical method
The statistical program SPSS 26.0 was used to analyze all of the data.The count data are shown as percentages (%).The chi-square (x2) test was conducted, and the results were fully analyzed.P <.05 was considered statistically significant.

CYP2C19 gene frequency and Hardy-Weinberg equilibrium
The frequencies of CYP2C19 polymorphic sites * 2 and * 3 in the patients in the current study were in accordance with the Hardy-Weinberg law of genetic equilibrium.These chosen objects are emblematic of their group (Table 2).

The frequency distribution of CYP2C19 genotypes, alleles and clopidogrel metabolic types in different sexes
Patients in this study were classified into a male group (343 males) and a female group (206 females).The results showed that there was no statistically significant difference in the frequency distribution of CYP2C19 genotypes between the two groups (x2=9.464,P=0.092).There was no statistically significant difference in the frequency distribution of CYP2C19 alleles between the two groups (x2=2.345,P=0.310).However, differences in the frequency distribution of clopidogrel metabolic types were statistically significant between the two groups (x2=7.544,P=0.023) (Table 3).4).

Logistic regression analysis
The possibilities of hyperglycemia, hypercholesterolemia, hypertriglyceridemia, hypo-HDL-cholesterolemia, hyper-LDL-cholesterolemia and high blood pressure were taken as the independent variables, while the possibility of clopidogrel resistance was taken as the dependent variable.In this study, wild-type CYP2C19*1/*1 was considered clopidogrel sensitive, and the other types were considered clopidogrel resistant.These 2 variables were substituted into the logistic regression equation.None of the independent variables were shown to be independent risk factors for clopidogrel resistance by logistic regression analysis (Table 5).
China is a country with a significant population of stroke patients, with approximately 3 million individuals experiencing their first ischaemic stroke every year [19].However, few studies have been conducted on the association between CYP2C19 gene polymorphisms and stroke.The associations between the CYP2C19 genotype distribution and three different types of stroke were also investigated in this work.Although the study preliminarily revealed no statistically significant associations among  these variables, the results need to be further studied due to the small number of patients who presented with cerebral embolism.According to the logistic regression analysis, extensive metabolizers were considered clopidogrel sensitive, and intermediate metabolizers and poor metabolizers were considered clopidogrel resistant.Some studies have proposed that the genotypes CYP2C19 *1/*2, *1/*3, *2/*2 and the CYP2C19 IM/ PM phenotypes may contribute to a heightened risk of developing hypertension [5].However, independent risk factors for clopidogrel resistance were not found in this analysis, which means that hyperglycaemia, hypercholesterolaemia, hypertriglyceridaemia, hypo-HDL-cholesterolaemia, hyper-LDL-cholesterolaemia and high blood pressure were not significantly associated with CYP2C19 genotype.
The intermediate metabolic type was the most common clopidogrel metabolic type in the Chaoshan district, accounting for 47.9%, while the poor metabolic type, accounting for 17.3%, was less common.In the Beijing district, the intermediate metabolic type accounted for 52.48%, and the poor metabolic type accounted for 9.90%.CYP2C19*3/*3 was not detected [36].In the Guizhou district, the most common metabolic type was the extensive metabolic type.The intermediate metabolic type accounted for 32.22%, and the poor metabolic type accounted for 10%.CYP2C19*3/*3 was also not detected [31].Only one CYP2C19*3/*3 patient was identified in this study, which to some extent indicates that this genotype has a lower frequency of distribution in the Chaoshan area.Although the most common metabolic type differed between Shantou and Guizhou, the least common type was the same in these three districts.Notably, the genotype CYP2C19*3/*3 is rare in these three districts.One study from Indonesia showed that the majority of patients were intermediate metabolizers, and only 2.4% of them had the homozygous mutant allele (*3/*3) [22].The most common diplotype in a Bulgarian psychiatric cohort was CYP2C19*1/*1 [14].In Vanessa et al. 's study, CYP2C19*1/*1 was also the most common phenotype among four cohorts, a subcohort of the Admixed American superpopulation from the One Thousand Genomes Project, HGDP Native Americans, and Kaingang and Guarani living in Brazil [9].
According to reports, those with the CYP2C19 allele with lower function are more likely to experience fatal complications and unfavourable cardiovascular events when treated with clopidogrel [16,35].Therefore, according to the Clinical Pharmacogenetics Implementation Consortium (CPIC), patients with CYP2C19 IM and PM phenotypes should receive other treatments, such as ticagrelor, an ADP receptor blocker that does not require enzymatic activation.Although ticagrelor has a greater risk of bleeding and lower patient adherence, it can inhibit platelet aggregation more quickly, to a greater extent, and more consistently [15,16,30].Thus, in the treatment of patients suffering from stroke, early CYP2C19 genotype testing and expedited reporting may be beneficial to patients, which is consistent with the opinion presented in another article [23] and may help neurologists provide more individualized precision therapy to patients.

Fig. 1
Fig. 1 Microarray image of the genotypes of CYP2C19

Table 2
CYP2C19 gene frequency and Hardy-Weinberg equilibrium

Table 3 .
The frequency distribution of CYP2C19 genotypes, alleles and clopidogrel metabolic types in different sexes

Table 4 .
The frequency distribution of CYP2C19 genotypes, alleles and clopidogrel metabolic types for different types of stroke

Table . 5
Logistic regression analysis